The use of RNA interference (RNAi) to silence the expression of specific genes associated with disease is one of the most promising new therapeutic paradigms in medicine. Although RNAi, and specifically small interfering RNA (siRNA), has shown much promise for the treatment of cancer and a broad spectrum of other diseases, delivery of RNAi is a challenge due to the vulnerability of RNA to serum nucleases, the propensity to cause off- target effects, and the requirement for delivery into the cytosol of target cells. Thus, new methods and vectors are needed to effectively deliver RNAi to target tissue. The objective of this proposal is to develop a new approach for targeted delivery of siRNA that capitalizes on the unique bioeffects that result from ultrasound (US)-induced vibrations of microbubles (MB) carrying siRNA. These bioeffects include enhanced cell membrane permeability to macromolecules, which is thought to facilitate siRNA internalization. We have developed an acoustically active cationic lipid MB carrier of EGFR siRNA that retards growth of murine squamous cell carcinomas. We have also recently designed a novel submicron polymer MB carrying EGFR siRNA-loaded liposomes that silences EGFR expression in vitro, and may have unique potential to facilitate extravascular siRNA transfer. Accordingly, we will test the overall hypothesis that siRNA delivery, gene silencing, and therapeutic effects can be achieved by optimal combinations of these 2 MB formulations and US parameters, using in vivo and in vitro models of squamous cell carcinoma as the test system. Four Aims are proposed: (1) To test the hypothesis that the loading efficiency of siRNA on MB and target cellular uptake can be increased by manipulation of MB chemistry and US parameters, we will experimentally determine the loading and cellular internalization of siRNA using a matrix of MB/US combinations (platforms) in cell culture. (2) To test the hypothesis that our US-MB siRNA delivery platform induces specific gene silencing, we will determine levels of EGFR silencing in vitro (and assess toxicity) using platforms from Aim 1. (3) To test the hypothesis tha our US-MB siRNA theranostics approach induces therapeutic gene silencing in vivo and that the RNAi will suppress tumor growth with favorable toxicity and biodistribution, we will determine EGFR silencing at a variety of doses in tumor bearing mice, assay tumor growth inhibition upon EGFR siRNA treatment, and determine biodistribution and toxicity profiles. (4) To investigate the mechanisms of US and MB mediated siRNA delivery, we will microscopically observe the trafficking of labeled siRNA, focusing on endocytotic and endocytosis-independent mechanisms. These studies will culminate in a non-invasive, targeted siRNA delivery strategy that will facilitate the clinical implementation of RNAi. Importantly, while our proposed siRNA delivery platform targets mRNA (and subsequent protein levels) of a specific oncogene, our work will establish general principles that can be extended to US-MB siRNA platforms for image-guided targeted gene silencing in other diseases for which specific gene silencing represents a therapeutic approach.